Objective: To determine the potential of opportunistic glycated haemoglobin (HbA1c) testing of pathology samples to detect previously unknown diabetes. Design: Pathology samples from participants collected for other reasons and suitable for HbA1c testing were utilised for opportunistic diabetes screening. HbA1c was measured with a Biorad Variant II turbo analyser and HbA1c levels of ≥6.5% (48 mmol/mol) were considered diagnostic for diabetes. Confirmation of previously unknown diabetes status was obtained by a review of hospital medical records and phone calls to general practitioners. Setting: Hospital pathology laboratory receiving samples from hospital-based and community-based (CB) settings. Participants: Participants were identified based on the blood sample collection location in the CB, emergency department (ED) and inpatient (IP) groups. Exclusions pretesting were made based on the electronic patient history of: age <18 years, previous diabetes diagnosis, query for diabetes status in the past 12 months, evidence of pregnancy and sample collected postsurgery or transfusion. Only one sample per individual participant was tested. Results: Of the 22 396 blood samples collected, 4505 (1142 CB, 1113 ED, 2250 IP) were tested of which 327 (7.3%) had HbA1c levels ≥6.5% (48 mmol/mol). Of these 120 (2.7%) were determined to have previously unknown diabetes (11 (1%) CB, 21 (1.9%) ED, 88 (3.9%) IP). The prevalence of previously unknown diabetes was substantially higher (5.4%) in hospital-based (ED and IP) participants aged over 54 years. Conclusions: Opportunistic testing of referred pathology samples can be an effective method of screening for diabetes, especially in hospital-based and older persons.